1. Field of the Invention (Technical Field)
The present invention relates to an apparatus and methods for controlled electrochemical release of DNA and other nucleic acids from electrodes, for use in cellular transfection, including gene therapy, and for other applications of controlled release of DNA and other nucleic acids.
2. Background Art
There is significant interest in controlled gene delivery for gene therapy in treatment of a wide variety of genetic diseases. In one mode of effecting gene therapy, macromolecules, generally DNA, are introduced into the cell. The cells are then transfected with the DNA, leading to transformed viable cells. Normal and functional genetic material is introduced into cells to correct an abnormality due to a defective or deficient gene product. Typically, the transferred genetic material contains the desired gene and a promoter to control the expression of the gene.
Nucleic acids as therapeutically effective substances are also used to inhibit specific cell functions. For example, antisense RNA and DNA sequences can be employed for selective inhibition of specific gene sequences. The antisense sequences act by blocking the expression of certain genes (such as deregulated oncogenes or viral genes) within the cell. The efficacy of short sequence antisense oligonucleotides has been demonstrated, even at low or comparatively low concentrations within the cell.
While significant progress has been made in manipulating genes at a cellular level, gene therapy for treatment of disease is limited and not commercially developed. There are a number of factors limiting effective gene therapy, including the number and rate of transformed cells required for a therapeutic effect. Efficiencies of transfection and transformation are related, in part, to the mode of delivery of the genetic material to and into the cell.
There are a number of methods that have been developed for the delivery of genetic material to and into the cell. These include a variety of physical delivery methods. Methods of physical delivery methods include electroporation; cell bombardment by coated molecules (called a xe2x80x9cgene gunxe2x80x9d); use of chemicals to increase cell membrane permeability, such as detergents or polyethyleneglycol; and various lipid-based techniques, such as liposome-cell fusion.
Electroporation utilizes short, high voltage electrical pulses to produce a transient high permeability state in cell membranes. The cells are exposed to exogenous DNA in high electric fields, with transference of DNA through the transient permeable hydrophilic pores. Electroporation techniques are taught in a number of patent applications, including U.S. Pat. Nos. 5,019,034, 5,749,847, 5,869,326, 5,993,434, and 6,014,584. Related techniques are taught in, for example, U.S. Pat. Nos. 4,081,340 and 5,964,726.
Another approach in gene therapy has been to use a gene spliced into a virus, such as a retrovirus or adenovirus, as a vector to introduce the gene into the target cell. This approach, however, has significant limitations, including safety and efficiency. In some applications, the approach requires the multiplication of target cells in vitro and subsequent reintroduction of the transformed cells. Even where the virus is introduced by direct administration of viral particles, there are other limitations to the approach. The size of the gene that can be carried by the viral vector is limited, and the genetic material that can be incorporated is similarly limited to DNA. Thus viral delivery approaches cannot be utilized with other nucleotides. Modifications of use of viruses in gene therapy, including use of non-viral sequences that bind cell membrane receptors, have also been explored. See, for examples, U.S. Pat. Nos. 5,916,803 and 6,022,735 and patents and publications cited therein.
A variety of lipid-based techniques have been employed, including use of liposomes. These methods are generally described in U.S. Pat. Nos. 5,892,071 and 5,908,635 and patents and publications cited therein.
With each of the methods, and particularly the physical delivery methods, there is a need to have the DNA, RNA or other nucleotide in immediately proximity to the cell membrane to be transited.
The invention includes a method for controlled nucleotide release, in which nucleotides to be released are first adsorbed on to at least one first electrode. A second electrode is provided, and the first and second electrodes are immersed in an aqueous media in which the nucleotide is to be released. A negative electrical charge is then applied to the first electrode relative to the second electrode, and the resulting release of nucleotide is proportional to the relative electrical charge difference and length of persistence of the charge difference. In this method, more than one first electrode may be employed, so that there may be two or more electrodes to which nucleotides are adsorbed, with one second electrode.
The step of adsorbing can be facilitated by applying a positive potential to the first electrode relative to the second electrode. Alternatively, the nucleotide may be adsorbed by incubation of the electrode in a solution containing the nucleotides for a suitable period. In the method, a third electrode can also be provided, where the second electrode serves as a counter electrode, and the third electrode serves as a reference electrode. In a preferred embodiment, the negative electrical charge is a potential difference of from about xe2x88x920.02 V to about xe2x88x921.4 V relative to the second electrode. The negative electrical charge may be pulsed, thereby effecting incremental release of the nucleotide
The nucleotides may be in a nucleotide and lipid complex, and the lipid may be a liposome. Similarly, the nucleotide may be thiolated. The nucleotide may be a single-stranded DNA, double-stranded DNA, RNA or other nucleic acid, and may also be an antisense nucleotide.
Also provided is a method for transporting nucleotides across tissue, in which nucleotides to be released are adsorbed on a first electrode. A second and third electrode are provided, and the second and third electrodes, together with the first electrode, are positioned in proximity to a region of tissue in an acqueous media in which the nucleotide is to be released. The tissue can include cells and cell membranes, and in the case of cells may be a monolayer or multiple layer of cells. A negative electrical charge is provided to the first electrode relative to the second electrode, and thereafter at least one electrical pulse is applied to the third electrode of sufficient voltage and duration to cause electroporation in the region of the tissue. In this way, the release of the nucleotide is proportional to the relative electrical charge difference and length of persistence of the charge difference and electroporation is related to the voltage and duration of the electrical pulse, so that the nucleotides are released and transported across the tissue. In the method, the adsorbing can include application of a positive potential to the first electrode to facilitate adsorption. The negative electrical charge applied to the first electrode relative to the second electrode may be a potential difference of from about xe2x88x920.02 V to about xe2x88x921.4 V. In this method, the negative electrical charge to the first electrode can be pulsed, thereby effecting incremental release of the nucleotide. Further, the electrical pulse to the third electrode, which provides for electroporation, may also be pulsed, and may further be pulsed in synchronicity with the negative electrical charge pulse.
In this method also the nucleotide may be thiolated, and may be a single-stranded DNA, double-stranded DNA RNA or other nucleic acid. The nucleotide may also be an antisense nucleotide.
The invention further includes an apparatus for controlled nucleotide release, the apparatus including a first electrode with a nucleotide adsorbed onto at least a portion of the surface, a second electrode, and a first power supply connected to the first and second electrode, providing a selectable positive or negative potential difference to the first electrode relative to the second electrode. In another embodiment, the apparatus can further include a third electrode and a second power supply connected to the second and third electrode, providing a selectable voltage output. In this apparatus, the first power supply provides a positive to negative potential difference to the first electrode relative to the second electrode from about at least +0.5 V to about at least xe2x88x921.4 V. The apparatus can also include a first pulse generator connected to the first power supply, capable of providing defined pulses of positive or negative potential difference to the first electrode relative to the second electrode. In the apparatus, it is preferred that the selectable voltage output of the second power supply is sufficient to cause electroporation of tissue which is placed in proximity to the second and third electrodes. The apparatus can also include a second pulse generator connected to the second power supply, and capable of providing defined pulses of the selectable voltage output.
In this apparatus, the nucleotides may be in a nucleotide and lipid complex, and the lipid may be a liposome. Similarly, the nucleotide may be thiolated. The nucleotide may be a single-stranded DNA, double-stranded DNA, RNA or other nucleic acid, and may also be an antisense nucleotide.
A primary object of the present invention is to provide a means for controlled and specific release of DNA and other nucleic acids within an aqueous environment, including within an organism or within cell culture media.
Another object of the present invention is to provide an apparatus and means for electrochemical immobilization of DNA and other nucleic acids on an electrode, and subsequent potential-controlled electrochemical release of the DNA or other nucleic acids from the electrode.
Another object of the present invention is to provide a non-viral method for delivery of DNA and other nucleic acids in close proximity to target cells, and subsequent release in conjunction with means for enhancing introduction of the DNA or other nucleic acids into the cell.
Another object of the present invention is to provide a two- or three-electrode system, whereby the amount and rate of release of immobilized DNA or other nucleic acids can be manipulated by electrical potential control.
Another object of the present invention is to provide a method for controlled and specific release of DNA and other nucleic acids for use in gene therapy for treatment or prevention of disease.
Another object of the present invention is to provide a means for electrochemical immobilization of either thiolated or non-thiolated DNA and other nucleic acids on an electrode, and subsequent potential-controlled electrochemical release of the thiolated or non-thiolated DNA or other nucleic acids.
Another object of the present invention is to provide a method for the controlled and specific release of complexes formed of lipids or liposomes and DNA or other nucleic acids for use in gene therapy for treatment or prevention of disease.
A primary advantage of the present invention is that it provides a method and apparatus for the controlled and specific release of DNA and other nucleic acids for use in gene therapy for treatment or prevention of disease.
Another advantage of the present invention is that electrochemical release of DNA or other nucleic acids may be combined with electroporation, so that a determined quantity of DNA or other nucleic acids may be released, and the DNA or other nucleic acids thereafter introduced into the target cell.
Another advantage of the present invention is that it permits delivery of DNA or other nucleic acids into a cellular target by means of either simultaneous or sequential electroporation, or by means of lipids or liposome complexes which transit the cellular membrane.
A further advantage of the present invention is that it provides a method for controlled electrochemical release of DNA or other nucleic acids and lipid complexes, which complexes may transit cell membranes and thereafter effect transformation of the target cell.
Other objects, advantages and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.